Members of the spotted fever group (SFG) of the genus Rickettsia are obligate intracellular bacteria that cause serious human illnesses such as Rocky Mountain Spotted Fever. They require the host actin cytoskeleton to facilitate internalization into host cells, and to power intracytoplasmic movement that enables spread between cells during infection. Therefore, elucidating how SFG Rickettsia manipulate the actin cytoskeleton is critical for understanding the process of pathogenesis. In addition, determining the mechanism of actin-based motility is of great interest from a cell biological perspective because actin filaments in Rickettsia comet tails differ considerably in their organization compared to filaments assembled by well-studied pathogens such as Listeria monocytogenes, suggesting that Rickettsia use different molecules and mechanisms to promote motility. Despite the importance of actin in entry and motility, very little is known about the molecular mechanisms used by the bacteria to manipulate the cytoskeleton. We recently identified a Rickettsia protein called RickA that is conserved among SFG species and stimulates actin polymerization by activating the host Arp2/3 complex, providing the first molecular insight into how Rickettsia harness actin. We now need to answer important questions about the role of RickA and Arp2/3 in infection. For example, do RickA and Arp2/3 complex play a functionally important role, and how to they act? When and how is RickA introduced into host cells to stimulate actin polymerization? What other cytoskeletal proteins are required to polymerize and organize actin filaments? Based our preliminary results, we propose a unifying hypothesis that RickA is translocated by bacteria into host cells using a type IV secretion system, where it activates Arp2/3 complex to initiate actin assembly during entry and/or motility. We further hypothesize that, during actin-based motility, RickA and Arp2/3 act transiently, and other cyoskeletal proteins are subsequently needed to generate the unique organization of actin filaments in comet tails. To test this hypothesis, we propose the following aims: (1) Determine the timing and localization of the secreted RickA protein and the mechanism of secretion, (2) Examine the function of Arp2/3 complex during entry and actin- based motility, and (3) test the role of the full spectrum of actin cytoskeletal proteins in entry and motility. Determining the mechanisms used by SFG Rickettsiae to manipulate the host actin cytoskeleton will shed light on an essential and poorly understood aspect of Rickettsia pathogenesis, and will also illuminate the mechanisms used by host cells to regulate the functions of the cytoskeleton. In the long-term these studies may result in the discovery of new paradigms for understanding host-pathogen interactions, and new approaches to diagnose and treat infections. [unreadable] [unreadable] [unreadable]